MOLECULAR MEDICINE REPORTS 12: 4123-4132, 2015
Chitosan as an adjuvant for a Helicobacter pylori therapeutic vaccine YANFENG GONG1*, LIMING TAO2*, FUCAI WANG1*, WEI LIU1, LEI JING1, DONGSHENG LIU1, SIJUN HU1, YONG XIE1 and NANJIN ZHOU3 Departments of 1Gastroenterology and 2Obstetrics, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330000; 3Department of Biochemistry and Molecular Biology, Jiangxi Medical Science Institute, Nanchang, Jiangxi 330006, P.R. China Received August 28, 2014; Accepted May 13, 2015 DOI: 10.3892/mmr.2015.3950 Abstract. The aim of the present study was to delineate the therapeutic effect of a Helicobacter pylori vaccine with chitosan as an adjuvant, as well as to identify the potential mechanism against H. pylori infection when compared with an H. pylori vaccine, with cholera toxin (CT) as an adjuvant. Mice were first infected with H. pylori and, following the establishment of an effective infection model, were vaccinated using an H. pylori protein vaccine with chitosan as an adjuvant. Levels of H. pylori colonization, H. pylori‑specific antibodies and cytokines were determined by enzyme‑linked immunosorbent assay. The TLR4 and Foxp3 mRNA and protein levels were determined by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. It was identified that the H. pylori elimination rate of the therapeutic vaccine with chitosan as an adjuvant (58.33%) was greater than the therapeutic vaccine with CT as an adjuvant (45.45%). The therapeutic H. pylori vaccine with chitosan as an adjuvant induced significantly greater antibody and cytokine levels when compared with the control groups. Notably, the IL‑10 and IL‑4 levels in the groups with chitosan as an adjuvant to the H. pylori vaccine were significantly greater than those in the groups with CT as an adjuvant. The mRNA expression levels of TLR4 and Foxp3 were significantly elevated in the mice that were vaccinated with chitosan as an adjuvant to the H. pylori vaccine,
Correspondence to: Dr Yong Xie, Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, 17 Yongwai Zheng Street, Nanchang, Jiangxi 330000, P.R. China E‑mail: [email protected]
Dr Nanjin Zhou, Department of Biochemistry and Molecular Biology, Jiangxi Medical Science Institute, 461 Bayi Road, Nanchang, Jiangxi 330006, P.R. China E‑mail: [email protected] *
Contributed equally
Key words: chitosan, Helicobacter pylori, vaccine, adjuvant
particularly in mice where the H. pylori infection had been eradicated. The H. pylori vaccine with chitosan as an adjuvant effectively increased the H. pylori elimination rate, the humoral immune response and the Th1/Th2 cell immune reaction; in addition, the therapeutic H. pylori vaccine regulated the Th1 and Th2 response. The significantly increased TLR4 expression and decreased CD4+CD25+Foxp3+Treg cell number contributed to the immune clearance of the H. pylori infection. Thus, the present findings demonstrate that in mice the H. pylori vaccine with chitosan as an adjuvant exerts an equivalent immunotherapeutic effect on H. pylori infection when compared with the H. pylori vaccine with CT as an adjuvant. Introduction Epidemiological evidence has indicated a highly significant association between the Helicobacter pylori infection and the development of duodenal ulcers and distal gastric adenocarcinoma. In 1994, H. pylori was categorized as a class I carcinogen/definite human carcinogen by the World Health Organization (1). Current antibiotic‑based therapeutic methods are not practical for global control (2), therefore, vaccines against the H. pylori infection are those that were developed in the past (3). H. pylori protein vaccines require an effective adjuvant (4) as H. pylori proteins exhibit a low immunogenicity, therefore, vaccination with an H. pylori antigen alone cannot induce a high enough immune response to deplete the Helicobacter infection and protect the gastric mucosa (5). Cholera toxin (CT) and heat‑labile Escherichia coli enterotoxin (LT) are generally regarded as the most powerful mucosal adjuvants (6,7); however, their use in humans is hampered by their particularly high toxicities. CT and LT have been restructured to reduce their toxicities (8), however this resulted in a reduction of their adjuvant effects. Chitosan, a polymer of D‑glucosamine and a natural product derived from chitin, is accessible, and demonstrates good bioadhesion, biodegradability and biocompatibility without immunogenicity, toxicity or side‑effects (9); thus, chitosan has been used in mucosal vaccines as an adjuvant (10). Numerous studies have indicated that chitosan effectively elicits a local (particularly mucosal local) immune
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response, enhances the ability of antigenic delivery systems and performs adjuvant activity in vaccines (11). It has been reported that Neisseria meningitidis and Bordetella pertussis vaccines with chitosan as the adjuvant successfully induced a protective immune response (12). Our previous study demonstrated that oral administration of H. pylori whole‑cell sonicate plus chitosan as the adjuvant protected mice against H. pylori infection (13). Furthermore, it has been shown that, as an adjuvant in vaccines for H. pylori protection, chitosan is more effective than CT in immune protection against H. pylori infection (14). However, to the best of our knowledge, there have been no reports regarding chitosan as an adjuvant for the H. pylori therapeutic vaccine and the immunoprotection mechanism remains unclear. Therefore, in the present study, mice were infected with H. pylori and then vaccinated using an H. pylori protein vaccine with chitosan as the adjuvant. This was to delineate the therapeutic effect of the H. pylori vaccine and the potential mechanism against H. pylori infection in comparison to a H. pylori vaccine with CT as the adjuvant. Materials and methods Reagents and bacterial strains. Chitosan and 88.5% deacetylated chitosan powder were purchased from Shanghai Qisheng Biological Preparation Co., Ltd. (Shanghai, China). Rabbit anti‑rat IgG1 (cat. no. PA1‑86329; Zymed Life Technologies, Carlsbad, CA, USA), IgG2a (cat. no. 61‑0220; Zymed Life Technologies) and IgA (cat. no. Sab3700520; Sigma‑Aldrich, St. Louis, MO, USA), and goat anti‑mouse IgG (cat. no. A27025; Zymed Life Technologies) peroxidase conjugate were purchased from Zymed Life Technologies (Carlsbad, CA, USA). CT was purchased from Sigma‑Aldrich. Enzyme‑linked immunosorbent assay (ELISA) kits for interleukin (IL)‑2, interferons (IFNs), IL‑12, IL‑4, and IL‑10 were purchased from eBioscience, Inc. (San Diego, CA, USA). Polymerase chain reaction (PCR) primers were purchased from Shanghai Sheng Gong Biological Engineering Technology Service Co., Ltd. (Shanghai, China) Goat anti‑mouse TLR4 polyclonal antibody (cat. no. sc‑12511) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti‑rat Foxp3 polyclonal antibody (cat. no. bs‑10211R) was purchased from Beijing Bo Orson Biological Technology Co., Ltd., (Beijing, China) and the H. pylori Sydney strain 1 (SS1) was provided by the H. pylori Strain Pool (Chinese Centre for Disease Control, China). An 450 enzyme microplate reader was purchased from Bio‑Rad Laboratories, Inc. (Hercules, CA, USA). A PCR thermal cycler was purchased from PerkinElmer, Inc. (Waltham, MA, USA). A JS680C gel imaging analysis system was purchased from Shanghai Peiqing Science and Technology Co., Ltd (Shanghai, China) and the ECP3000 electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory (Beijing, China). A BH‑2 stereo‑binocular microscope was purchased from Suzhou REIT Image Technology Co., Ltd. (China). Animals. Female BALB/c mice (age, 6‑8 weeks; mean weight, 22.5 g) were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed
in a specific pathogen‑free environment with free access to food and water. All animal experiments were conducted in accordance with principles stated in the Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University. H. pylori culture. The H. pylori SS1 was used throughout the investigation. H. pylori was grown in a Campylobacter agar base, containing 10% sheep blood, under microaerobic conditions (5% O2, 10% CO2 and 85% N2) at 37˚C for 2‑3 days. Preparation of H. pylori antigen. After culturing for 2‑3 days, the H. pylori SS1 was eluted with phosphate‑buffered saline (PBS) and centrifuged at 10,000 x g and 4˚C for 10 min. The pellet was washed and sonicated. Following centrifugation at 8,000 x g and 4˚C for 30 min, the supernatant was collected and stored at ‑80˚C until use. The protein concentration was determined using a bicinchoninic acid (BCA) assay. Preparation of chitosan particles and solution. Deacetylated (88.5%) chitosan powder was suspended in saline to a final concentration of 10 mg/ml and sonicated twice (output, 80 Hz). The small particles in the supernatant were removed. The chitosan particles were collected by further centrifugation at 140 x g for 10 min. Chitosan stock solution [3% (w/w)] was prepared from 88.5% deacetylated chitosan powder in 0.8% (v/v) acetic acid and 0.9% (w/v) saline. H. pylori infection. Each mouse was orally administered with 1x109 colony‑forming units (CFUs) of H. pylori per liter five times every other day. Twelve weeks after the last inoculation, four mice were euthanized, and the stomachs were removed to ascertain whether the H. pylori infection model had been established. H. pylori vaccination. The infected BALB/c mice were orally immunized in the following groups at days 0, 7, 14 and 21: i) Control (PBS alone), 12 mice; ii) H. pylori antigen alone, 11 mice; iii) H. pylori antigen plus 0.5% chitosan solution, 12 mice; iv) H. pylori antigen plus CT (5 µg/mouse), 11 mice (one mouse died); and v) H. pylori antigen plus chitosan particles (500 µg/mouse), 12 mice. Four weeks after the final vaccination, saliva and blood samples were collected. The stomachs were isolated and cut longitudinally into two sections. One was used for examination of H. pylori, and the other was used for histology and immunological assays. Assessment of bacterial load in the stomach. The bacterial load in the stomach was determined by quantitative culture of H. pylori and Giemsa staining. H. pylori‑negative was defined when the culture of H. pylori and the Giemsa staining were negative. H. pylori‑positive was defined when either the culture of H. pylori or the Giemsa staining was positive. For assessment of H. pylori colonization, the weighed stomachs were homogenized in Brucella broth and spread over a serum plate. The plates were incubated for 3‑7 days, and the H. pylori colonies were counted and calculated as CFUs per gram of stomach tissue. For Giemsa staining, the colonization was assessed by semi‑quantitative analysis of H. pylori in the
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B
Figure 1. (A) Giemsa staining of the gastric mucosa (magnification, x400). Mice were euthanized 12 weeks after the H. pylori inoculation and the tissue sections were stained by Giemsa staining. The red circle indicates H. pylori colonization in the gastric pits. (B) Hematoxylin and eosin staining of the gastric mucosa (magnification, x400). Mice were euthanized 12 weeks after the H. pylori inoculation. The red arrow indicates mucosal hyperemia, lymphocytes and neutrophil infiltration.
gastric mucosa (0, nil; 1, 1‑2 cells/crypt; 2, 3‑10 cells/crypt; 3, 11‑20 cells/crypt; and 4, >21 cells/crypt). Determination of H. pylori‑specific antibody levels in the gastric mucosa and saliva. The H. pylori‑specific antibodies, IgG, IgG1, and IgG2a in sera, and IgA in the gastric mucosa and saliva were detected by indirect ELISA. After weighing, the gastric mucosa was homogenized in PBS and the homogenates were centrifuged at 3,000 x g at 4˚C for 20 min. The supernatant was harvested and diluted at 1:2. The sera and saliva were diluted at 1:100 and 1:5, respectively. Peroxidase‑conjugated rabbit anti‑rat IgG1, IgG2a or IgA was diluted at 1:1,000 and peroxidase‑conjugated goat anti‑mouse IgG secondary antibody was diluted at 1:2,000. The antibody levels of each immunized group from the sera and saliva were represented as relative levels to the mock‑immunized control group. The IgA levels in the gastric mucosa were represented as relative levels (per gram wet weight of the gastric mucosa) to the mock‑immunized group. Determination of cytokines in the gastric mucosa by ELISA. After weighing, the gastric mucosa was homogenized in PBS and the homogenates were centrifuged at 3,000 x g at 4˚C for 20 min. ELISA kits were used to quantify IL‑2, IFN, IL‑12, IL‑4 and IL‑10 in the supernatants (diluted at 1:2) following centrifugation. The results were represented as pg/mg wet weight of the gastric mucosa. Determination of TLR4 and Foxp3 mRNA contents in the gastric mucosa by reverse transcription (RT)‑PCR. Total RNA was isolated from the mouse gastric mucosa to determine TLR4 and Foxp3 mRNA levels within the gastric mucosa using RT‑PCR. The cDNA from each sample served as a template for subsequent PCR assays to assess the TLR4 and Foxp3 mRNA levels, which were normalized to the expression of β‑actin. Each 50‑µl PCR consisted of 25 pmol of each primer, 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 200 µM dNTPs, and 0.5 µl of Taq enzyme. β‑actin and TLR4 were amplified at 95˚C for 5 min (1 cycle); 95˚C for 30 sec, 56˚C for 30 sec and 72˚C for 1 min (30 cycles); and 72˚C for 5 min (1 cycle).
Foxp3 was amplified at 95˚C for 2.5 min (1 cycle); 95˚C for 30 sec, 57˚C for 30 sec and 72˚C for 1 min (32 cycles); 72˚C for 5 min (1 cycle). The PCR products were visualized following electrophoresis on 2% agarose gels and the band intensities were quantified by densitometry. Determination of TLR4 and Foxp3 protein expression in the gastric mucosa by immunohistochemistry. Snap‑frozen biopsies were cut into 4‑µm sections to determine the levels of TLR4 and Foxp3 protein expression in the gastric mucosa by immunohistochemistry. In the TLR4‑stained sections, positive cells were assigned to the cell membrane or to yellow/brown‑dyed plasma, and negative cells were assigned to the cell membrane or to non‑dyed plasma. In Foxp3‑stained sections, positive cells were assigned to the cell nucleus or to yellow/brown‑dyed plasma and negative cells were assigned to the cell membrane or to non‑dyed plasma. The immunoreaction was graded according to the depth of the color and the proportion of positive cells. The degree of dyeing was divided into the following score grades and expressed as a percentage: 0, Negative; 1, (yellow) weakly positive; 2 (light brown) positive; and 3 (brown) strongly positive. Statistical analysis. Differences in the eradication rate were analyzed by Fisher's exact test. Differences in H. pylori‑specific antibody levels in the gastric mucosa among the experimental groups were evaluated for statistical significance by analysis of variance or Student's t‑test. P
Chitosan as an adjuvant for a Helicobacter pylori therapeutic vaccine YANFENG GONG1*, LIMING TAO2*, FUCAI WANG1*, WEI LIU1, LEI JING1, DONGSHENG LIU1, SIJUN HU1, YONG XIE1 and NANJIN ZHOU3 Departments of 1Gastroenterology and 2Obstetrics, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi 330000; 3Department of Biochemistry and Molecular Biology, Jiangxi Medical Science Institute, Nanchang, Jiangxi 330006, P.R. China Received August 28, 2014; Accepted May 13, 2015 DOI: 10.3892/mmr.2015.3950 Abstract. The aim of the present study was to delineate the therapeutic effect of a Helicobacter pylori vaccine with chitosan as an adjuvant, as well as to identify the potential mechanism against H. pylori infection when compared with an H. pylori vaccine, with cholera toxin (CT) as an adjuvant. Mice were first infected with H. pylori and, following the establishment of an effective infection model, were vaccinated using an H. pylori protein vaccine with chitosan as an adjuvant. Levels of H. pylori colonization, H. pylori‑specific antibodies and cytokines were determined by enzyme‑linked immunosorbent assay. The TLR4 and Foxp3 mRNA and protein levels were determined by reverse transcription polymerase chain reaction and immunohistochemistry, respectively. It was identified that the H. pylori elimination rate of the therapeutic vaccine with chitosan as an adjuvant (58.33%) was greater than the therapeutic vaccine with CT as an adjuvant (45.45%). The therapeutic H. pylori vaccine with chitosan as an adjuvant induced significantly greater antibody and cytokine levels when compared with the control groups. Notably, the IL‑10 and IL‑4 levels in the groups with chitosan as an adjuvant to the H. pylori vaccine were significantly greater than those in the groups with CT as an adjuvant. The mRNA expression levels of TLR4 and Foxp3 were significantly elevated in the mice that were vaccinated with chitosan as an adjuvant to the H. pylori vaccine,
Correspondence to: Dr Yong Xie, Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, 17 Yongwai Zheng Street, Nanchang, Jiangxi 330000, P.R. China E‑mail: [email protected]
Dr Nanjin Zhou, Department of Biochemistry and Molecular Biology, Jiangxi Medical Science Institute, 461 Bayi Road, Nanchang, Jiangxi 330006, P.R. China E‑mail: [email protected] *
Contributed equally
Key words: chitosan, Helicobacter pylori, vaccine, adjuvant
particularly in mice where the H. pylori infection had been eradicated. The H. pylori vaccine with chitosan as an adjuvant effectively increased the H. pylori elimination rate, the humoral immune response and the Th1/Th2 cell immune reaction; in addition, the therapeutic H. pylori vaccine regulated the Th1 and Th2 response. The significantly increased TLR4 expression and decreased CD4+CD25+Foxp3+Treg cell number contributed to the immune clearance of the H. pylori infection. Thus, the present findings demonstrate that in mice the H. pylori vaccine with chitosan as an adjuvant exerts an equivalent immunotherapeutic effect on H. pylori infection when compared with the H. pylori vaccine with CT as an adjuvant. Introduction Epidemiological evidence has indicated a highly significant association between the Helicobacter pylori infection and the development of duodenal ulcers and distal gastric adenocarcinoma. In 1994, H. pylori was categorized as a class I carcinogen/definite human carcinogen by the World Health Organization (1). Current antibiotic‑based therapeutic methods are not practical for global control (2), therefore, vaccines against the H. pylori infection are those that were developed in the past (3). H. pylori protein vaccines require an effective adjuvant (4) as H. pylori proteins exhibit a low immunogenicity, therefore, vaccination with an H. pylori antigen alone cannot induce a high enough immune response to deplete the Helicobacter infection and protect the gastric mucosa (5). Cholera toxin (CT) and heat‑labile Escherichia coli enterotoxin (LT) are generally regarded as the most powerful mucosal adjuvants (6,7); however, their use in humans is hampered by their particularly high toxicities. CT and LT have been restructured to reduce their toxicities (8), however this resulted in a reduction of their adjuvant effects. Chitosan, a polymer of D‑glucosamine and a natural product derived from chitin, is accessible, and demonstrates good bioadhesion, biodegradability and biocompatibility without immunogenicity, toxicity or side‑effects (9); thus, chitosan has been used in mucosal vaccines as an adjuvant (10). Numerous studies have indicated that chitosan effectively elicits a local (particularly mucosal local) immune
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response, enhances the ability of antigenic delivery systems and performs adjuvant activity in vaccines (11). It has been reported that Neisseria meningitidis and Bordetella pertussis vaccines with chitosan as the adjuvant successfully induced a protective immune response (12). Our previous study demonstrated that oral administration of H. pylori whole‑cell sonicate plus chitosan as the adjuvant protected mice against H. pylori infection (13). Furthermore, it has been shown that, as an adjuvant in vaccines for H. pylori protection, chitosan is more effective than CT in immune protection against H. pylori infection (14). However, to the best of our knowledge, there have been no reports regarding chitosan as an adjuvant for the H. pylori therapeutic vaccine and the immunoprotection mechanism remains unclear. Therefore, in the present study, mice were infected with H. pylori and then vaccinated using an H. pylori protein vaccine with chitosan as the adjuvant. This was to delineate the therapeutic effect of the H. pylori vaccine and the potential mechanism against H. pylori infection in comparison to a H. pylori vaccine with CT as the adjuvant. Materials and methods Reagents and bacterial strains. Chitosan and 88.5% deacetylated chitosan powder were purchased from Shanghai Qisheng Biological Preparation Co., Ltd. (Shanghai, China). Rabbit anti‑rat IgG1 (cat. no. PA1‑86329; Zymed Life Technologies, Carlsbad, CA, USA), IgG2a (cat. no. 61‑0220; Zymed Life Technologies) and IgA (cat. no. Sab3700520; Sigma‑Aldrich, St. Louis, MO, USA), and goat anti‑mouse IgG (cat. no. A27025; Zymed Life Technologies) peroxidase conjugate were purchased from Zymed Life Technologies (Carlsbad, CA, USA). CT was purchased from Sigma‑Aldrich. Enzyme‑linked immunosorbent assay (ELISA) kits for interleukin (IL)‑2, interferons (IFNs), IL‑12, IL‑4, and IL‑10 were purchased from eBioscience, Inc. (San Diego, CA, USA). Polymerase chain reaction (PCR) primers were purchased from Shanghai Sheng Gong Biological Engineering Technology Service Co., Ltd. (Shanghai, China) Goat anti‑mouse TLR4 polyclonal antibody (cat. no. sc‑12511) was purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Rabbit anti‑rat Foxp3 polyclonal antibody (cat. no. bs‑10211R) was purchased from Beijing Bo Orson Biological Technology Co., Ltd., (Beijing, China) and the H. pylori Sydney strain 1 (SS1) was provided by the H. pylori Strain Pool (Chinese Centre for Disease Control, China). An 450 enzyme microplate reader was purchased from Bio‑Rad Laboratories, Inc. (Hercules, CA, USA). A PCR thermal cycler was purchased from PerkinElmer, Inc. (Waltham, MA, USA). A JS680C gel imaging analysis system was purchased from Shanghai Peiqing Science and Technology Co., Ltd (Shanghai, China) and the ECP3000 electrophoresis apparatus was purchased from Beijing Liuyi Instrument Factory (Beijing, China). A BH‑2 stereo‑binocular microscope was purchased from Suzhou REIT Image Technology Co., Ltd. (China). Animals. Female BALB/c mice (age, 6‑8 weeks; mean weight, 22.5 g) were purchased from the Animal Center of the Chinese Academy of Sciences (Shanghai, China). The mice were housed
in a specific pathogen‑free environment with free access to food and water. All animal experiments were conducted in accordance with principles stated in the Guide for the Care and Use of Laboratory Animals. The experimental protocols were approved by the Ethics Committee of The First Affiliated Hospital of Nanchang University. H. pylori culture. The H. pylori SS1 was used throughout the investigation. H. pylori was grown in a Campylobacter agar base, containing 10% sheep blood, under microaerobic conditions (5% O2, 10% CO2 and 85% N2) at 37˚C for 2‑3 days. Preparation of H. pylori antigen. After culturing for 2‑3 days, the H. pylori SS1 was eluted with phosphate‑buffered saline (PBS) and centrifuged at 10,000 x g and 4˚C for 10 min. The pellet was washed and sonicated. Following centrifugation at 8,000 x g and 4˚C for 30 min, the supernatant was collected and stored at ‑80˚C until use. The protein concentration was determined using a bicinchoninic acid (BCA) assay. Preparation of chitosan particles and solution. Deacetylated (88.5%) chitosan powder was suspended in saline to a final concentration of 10 mg/ml and sonicated twice (output, 80 Hz). The small particles in the supernatant were removed. The chitosan particles were collected by further centrifugation at 140 x g for 10 min. Chitosan stock solution [3% (w/w)] was prepared from 88.5% deacetylated chitosan powder in 0.8% (v/v) acetic acid and 0.9% (w/v) saline. H. pylori infection. Each mouse was orally administered with 1x109 colony‑forming units (CFUs) of H. pylori per liter five times every other day. Twelve weeks after the last inoculation, four mice were euthanized, and the stomachs were removed to ascertain whether the H. pylori infection model had been established. H. pylori vaccination. The infected BALB/c mice were orally immunized in the following groups at days 0, 7, 14 and 21: i) Control (PBS alone), 12 mice; ii) H. pylori antigen alone, 11 mice; iii) H. pylori antigen plus 0.5% chitosan solution, 12 mice; iv) H. pylori antigen plus CT (5 µg/mouse), 11 mice (one mouse died); and v) H. pylori antigen plus chitosan particles (500 µg/mouse), 12 mice. Four weeks after the final vaccination, saliva and blood samples were collected. The stomachs were isolated and cut longitudinally into two sections. One was used for examination of H. pylori, and the other was used for histology and immunological assays. Assessment of bacterial load in the stomach. The bacterial load in the stomach was determined by quantitative culture of H. pylori and Giemsa staining. H. pylori‑negative was defined when the culture of H. pylori and the Giemsa staining were negative. H. pylori‑positive was defined when either the culture of H. pylori or the Giemsa staining was positive. For assessment of H. pylori colonization, the weighed stomachs were homogenized in Brucella broth and spread over a serum plate. The plates were incubated for 3‑7 days, and the H. pylori colonies were counted and calculated as CFUs per gram of stomach tissue. For Giemsa staining, the colonization was assessed by semi‑quantitative analysis of H. pylori in the
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Figure 1. (A) Giemsa staining of the gastric mucosa (magnification, x400). Mice were euthanized 12 weeks after the H. pylori inoculation and the tissue sections were stained by Giemsa staining. The red circle indicates H. pylori colonization in the gastric pits. (B) Hematoxylin and eosin staining of the gastric mucosa (magnification, x400). Mice were euthanized 12 weeks after the H. pylori inoculation. The red arrow indicates mucosal hyperemia, lymphocytes and neutrophil infiltration.
gastric mucosa (0, nil; 1, 1‑2 cells/crypt; 2, 3‑10 cells/crypt; 3, 11‑20 cells/crypt; and 4, >21 cells/crypt). Determination of H. pylori‑specific antibody levels in the gastric mucosa and saliva. The H. pylori‑specific antibodies, IgG, IgG1, and IgG2a in sera, and IgA in the gastric mucosa and saliva were detected by indirect ELISA. After weighing, the gastric mucosa was homogenized in PBS and the homogenates were centrifuged at 3,000 x g at 4˚C for 20 min. The supernatant was harvested and diluted at 1:2. The sera and saliva were diluted at 1:100 and 1:5, respectively. Peroxidase‑conjugated rabbit anti‑rat IgG1, IgG2a or IgA was diluted at 1:1,000 and peroxidase‑conjugated goat anti‑mouse IgG secondary antibody was diluted at 1:2,000. The antibody levels of each immunized group from the sera and saliva were represented as relative levels to the mock‑immunized control group. The IgA levels in the gastric mucosa were represented as relative levels (per gram wet weight of the gastric mucosa) to the mock‑immunized group. Determination of cytokines in the gastric mucosa by ELISA. After weighing, the gastric mucosa was homogenized in PBS and the homogenates were centrifuged at 3,000 x g at 4˚C for 20 min. ELISA kits were used to quantify IL‑2, IFN, IL‑12, IL‑4 and IL‑10 in the supernatants (diluted at 1:2) following centrifugation. The results were represented as pg/mg wet weight of the gastric mucosa. Determination of TLR4 and Foxp3 mRNA contents in the gastric mucosa by reverse transcription (RT)‑PCR. Total RNA was isolated from the mouse gastric mucosa to determine TLR4 and Foxp3 mRNA levels within the gastric mucosa using RT‑PCR. The cDNA from each sample served as a template for subsequent PCR assays to assess the TLR4 and Foxp3 mRNA levels, which were normalized to the expression of β‑actin. Each 50‑µl PCR consisted of 25 pmol of each primer, 10 mM Tris (pH 8.3), 1.5 mM MgCl2, 200 µM dNTPs, and 0.5 µl of Taq enzyme. β‑actin and TLR4 were amplified at 95˚C for 5 min (1 cycle); 95˚C for 30 sec, 56˚C for 30 sec and 72˚C for 1 min (30 cycles); and 72˚C for 5 min (1 cycle).
Foxp3 was amplified at 95˚C for 2.5 min (1 cycle); 95˚C for 30 sec, 57˚C for 30 sec and 72˚C for 1 min (32 cycles); 72˚C for 5 min (1 cycle). The PCR products were visualized following electrophoresis on 2% agarose gels and the band intensities were quantified by densitometry. Determination of TLR4 and Foxp3 protein expression in the gastric mucosa by immunohistochemistry. Snap‑frozen biopsies were cut into 4‑µm sections to determine the levels of TLR4 and Foxp3 protein expression in the gastric mucosa by immunohistochemistry. In the TLR4‑stained sections, positive cells were assigned to the cell membrane or to yellow/brown‑dyed plasma, and negative cells were assigned to the cell membrane or to non‑dyed plasma. In Foxp3‑stained sections, positive cells were assigned to the cell nucleus or to yellow/brown‑dyed plasma and negative cells were assigned to the cell membrane or to non‑dyed plasma. The immunoreaction was graded according to the depth of the color and the proportion of positive cells. The degree of dyeing was divided into the following score grades and expressed as a percentage: 0, Negative; 1, (yellow) weakly positive; 2 (light brown) positive; and 3 (brown) strongly positive. Statistical analysis. Differences in the eradication rate were analyzed by Fisher's exact test. Differences in H. pylori‑specific antibody levels in the gastric mucosa among the experimental groups were evaluated for statistical significance by analysis of variance or Student's t‑test. P
